The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Cytochrome P450 (P450, CYP) research provides many opportunities for the application of kinetic isotope effect (KIE) strategies. P450s collectively catalyze oxidations of more substrates than any other group of enzymes, and CH bond cleavage is a major feature in a large fraction of these reactions. The presence of a significant primary deuterium KIE is evidence that hydrogen abstraction is at least partially rate-limiting in the reactions, and this appears to be the case in many P450 reactions. The first report of a KIE in (P450-linked) drug metabolism appeared in 1961 (for morphine N-demethylation), and in a number of cases, it has been possible to modulate the in vivo metabolism or toxicity of chemicals by deuterium substitution. A number of efforts are in progress to utilize deuterium substitution to alter the metabolism of drugs in an advantageous manner.
© 2017 Elsevier Inc. All rights reserved.
Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern-Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K 1.8-2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K 0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.
Emerging techniques now allow for precise quantification of distributions of biological molecules in single cells. These rapidly advancing experimental methods have created a need for more rigorous and efficient modeling tools. Here, we derive new bounds on the likelihood that observations of single-cell, single-molecule responses come from a discrete stochastic model, posed in the form of the chemical master equation. These strict upper and lower bounds are based on a finite state projection approach, and they converge monotonically to the exact likelihood value. These bounds allow one to discriminate rigorously between models and with a minimum level of computational effort. In practice, these bounds can be incorporated into stochastic model identification and parameter inference routines, which improve the accuracy and efficiency of endeavors to analyze and predict single-cell behavior. We demonstrate the applicability of our approach using simulated data for three example models as well as for experimental measurements of a time-varying stochastic transcriptional response in yeast.
Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for free-solution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R(2)> 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.
Measurements of spin-lock relaxation rates in the rotating frame (R1ρ ) at high magnetic fields afford the ability to probe not only relatively slow molecular motions, but also other dynamic processes, such as chemical exchange and diffusion. In particular, measurements of the variation (or dispersion) of R1ρ with locking field allow the derivation of quantitative parameters that describe these processes. Measurements in deuterated solutions demonstrate the manner and degree to which exchange dominates relaxation at high fields (4.7 T, 7 T) in simple solutions, whereas temperature and pH are shown to be very influential factors affecting the rates of proton exchange. Simulations and experiments show that multiple exchanging pools of protons in realistic tissues can be assumed to behave independently of each other. R1ρ measurements can be combined to derive an exchange rate contrast (ERC) that produces images whose intensities emphasize protons with specific exchange rates rather than chemical shifts. In addition, water diffusion in the presence of intrinsic susceptibility gradients may produce significant effects on R1ρ dispersions at high fields. The exchange and diffusion effects act independently of each other, as confirmed by simulation and experimentally in studies of red blood cells at different levels of oxygenation. Collectively, R1ρ measurements provide an ability to quantify exchange processes, to provide images that depict protons with specific exchange rates and to describe the microstructure of tissues containing magnetic inhomogeneities. As such, they complement traditional T1 or T2 measurements and provide additional insights from measurements of R1ρ at a single locking field. Copyright © 2016 John Wiley & Sons, Ltd.
Copyright © 2016 John Wiley & Sons, Ltd.
(-)-Centratherin is a bioactive sesquiterpenoid lactone, whose absolute configuration (AC) was not established, but has been proposed based on those of germacrane precursors. To verify this proposal, the experimental electronic circular dichroism (ECD), electronic dissymmetry factor (EDF), optical rotatory dispersion (ORD), vibrational circular dichroism (VCD), and vibrational dissymmetry factor (VDF) spectra of (-)-centratherin have been analyzed with the corresponding density functional theoretical predictions. These analyses suggest the AC of naturally occurring (-)-centratherin to be (6R,7R,8S,10R,2'Z).
Models that capture the chemical kinetics of cellular regulatory networks can be specified in terms of rules for biomolecular interactions. A rule defines a generalized reaction, meaning a reaction that permits multiple reactants, each capable of participating in a characteristic transformation and each possessing certain, specified properties, which may be local, such as the state of a particular site or domain of a protein. In other words, a rule defines a transformation and the properties that reactants must possess to participate in the transformation. A rule also provides a rate law. A rule-based approach to modeling enables consideration of mechanistic details at the level of functional sites of biomolecules and provides a facile and visual means for constructing computational models, which can be analyzed to study how system-level behaviors emerge from component interactions.
Computational protein design has found great success in engineering proteins for thermodynamic stability, binding specificity, or enzymatic activity in a 'single state' design (SSD) paradigm. Multi-specificity design (MSD), on the other hand, involves considering the stability of multiple protein states simultaneously. We have developed a novel MSD algorithm, which we refer to as REstrained CONvergence in multi-specificity design (RECON). The algorithm allows each state to adopt its own sequence throughout the design process rather than enforcing a single sequence on all states. Convergence to a single sequence is encouraged through an incrementally increasing convergence restraint for corresponding positions. Compared to MSD algorithms that enforce (constrain) an identical sequence on all states the energy landscape is simplified, which accelerates the search drastically. As a result, RECON can readily be used in simulations with a flexible protein backbone. We have benchmarked RECON on two design tasks. First, we designed antibodies derived from a common germline gene against their diverse targets to assess recovery of the germline, polyspecific sequence. Second, we design "promiscuous", polyspecific proteins against all binding partners and measure recovery of the native sequence. We show that RECON is able to efficiently recover native-like, biologically relevant sequences in this diverse set of protein complexes.
A versatile and scalable method for fabricating shape-engineered nano- and micrometer scale particles from mesoporous silicon (PSi) thin films is presented. This approach, based on the direct imprinting of porous substrates (DIPS) technique, facilitates the generation of particles with arbitrary shape, ranging in minimum dimension from approximately 100 nm to several micrometers, by carrying out high-pressure (>200 MPa) direct imprintation, followed by electrochemical etching of a sub-surface perforation layer and ultrasonication. PSi particles (PSPs) with a variety of geometries have been produced in quantities sufficient for biomedical applications (≫10 μg). Because the stamps can be reused over 150 times, this process is substantially more economical and efficient than the use of electron beam lithography and reactive ion etching for the fabrication of nanometer-scale PSPs directly. The versatility of this fabrication method is demonstrated by loading the DIPS-imprinted PSPs with a therapeutic peptide nucleic acid drug molecule, and by vapor deposition of an Au coating to facilitate the use of PSPs as a photothermal contrast agent.
This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases.
Copyright © 2014 John Wiley & Sons, Inc.